从一个不活跃的 prokaryotic SOD 同类物到一个活跃的蛋白质通过位点定向突变发生
Lucia Banci1, Manuela Benvenuti, Ivano Bertini
1Department of Chemistry and Centro Risonanze Magnetiche, University of Florence, 50019 Sesto Fiorentino, Italy.
Journal of the American Chemical Society
|September 22, 2005
概括
研究人员通过重新引入铜联体来设计一种类似于SOD的Bacillus subtilis蛋白质. 经过修改的酶显示了部分活性,突出显示了特定金属协调对超氧化物脱酶功能的重要性.
科学领域:
- 生物化学 生物化学
- 结构生物学 结构生物学
- 酶学 是一种酶学.
背景情况:
- Prokaryotic Cu,ZnSOD 往往缺乏在真核生物对应物中发现的必不可少的金属连接体.
- 了解这些差异是解读酶功能和进化过程的关键.
研究的目的:
- 为了研究一种类似于SOD的Bacillus subtilis蛋白质中缺失的铜连接体的作用.
- 通过逐步重新引入金属结合点来设计一种功能性酶.
主要方法:
- 位点定向的突变发生,以重新引入铜配体.
- 生物化学测试用于测量酶活性.
- 溶液和晶体状态的表征 (例如,光谱学,X射线晶体学).
主要成果:
- 一个缺少一个铜配体的突变体是不活跃的.
- 与人类Cu,ZnSOD相比,一个完全复制的突变体表现出~10%的活性.
- 蛋白质在溶液中表现出形状障碍,但在晶体中表现出明确的结构.
结论:
- 特定的铜联体协调对于Cu,ZnSOD活动至关重要.
- 溶液中的 conformational 动力学可能会影响酶功能.
- 这项研究提供了对人工酶进化和SOD突变者行为的见解.
更多相关视频
10:31Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability
Published on: February 3, 2022
08:11Adaptation at the Extremes of Life: Experimental Evolution with the Extremophile Archaeon Sulfolobus acidocaldarius
Published on: June 14, 2024
相关概念视频
Mutations
Overview
From DNA to Protein
The flow of genetic information in cells from DNA to mRNA to protein is described by the central dogma, which states that genes specify the sequence of mRNAs, which in turn specify the sequence of amino acids making up all proteins. The decoding of one molecule to another is performed by specific proteins and RNAs. Because the information stored in DNA is so central to cellular function, it makes intuitive sense that the cell would make mRNA copies of this information for protein synthesis...
Conservative Site-specific Recombination and Phase Variation
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...
Mutations
Mutations are changes in the sequence of DNA. These changes can occur spontaneously or they can be induced by exposure to environmental factors. Mutations can be characterized in a number of different ways: whether and how they alter the amino acid sequence of the protein, whether they occur over a small or large area of DNA, and whether they occur in somatic cells or germline cells.
Chromosomal Alterations Are Large-Scale Mutations
While point mutations are changes in a single nucleotide in...
Chromosomal Alterations Are Large-Scale Mutations
While point mutations are changes in a single nucleotide in...
Replication in Prokaryotes
DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the single origin of replication (ori) on the cell's circular chromosome. Replication then proceeds around the entire circle of the chromosome in each direction from the two replication forks, resulting in two DNA molecules.
Many Proteins Work Together to Replicate the Chromosome
Replication is coordinated and carried out by a host of specialized...
Many Proteins Work Together to Replicate the Chromosome
Replication is coordinated and carried out by a host of specialized...
Bacterial Protein Maturation
Bacterial protein maturation is a tightly regulated process that ensures newly synthesized polypeptides achieve correct functional conformations. This maturation involves a series of modifications, folding events, and quality control steps, often assisted by specialized chaperone proteins.N-Terminal ModificationsThe maturation of bacterial polypeptides begins cotranslationally as the polypeptide exits the ribosome. The first amino acid, N-formylmethionine (fMet), is typically modified at the...
